JP2002154976A - Agent and health food for preventing, ameliorating and/ or treating chronic disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an agent and a health food effective for preventing, ameliorating and/or treating chronic diseases, persistently exhibiting effects for a long period of time. SOLUTION: This agent for preventing, ameliorating and/or treating chronic diseases is characterized in that the agent contains a bacterium recombined with a physiologically active substance gene and the recombinant bacterium can produce the physiologically active substance in an animal intestinal tract. This health food comprises the recombinant bacterium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel pharmaceutical or health food used for preventing, improving and / or treating chronic diseases such as lifestyle-related diseases. More specifically, the present invention relates to a novel pharmaceutical or health food capable of preventing, ameliorating, and / or treating the chronic disease by producing a bioactive substance in the intestinal tract of an animal using a genetically modified microorganism.

[0002]

2. Description of the Related Art Conventionally, as a drug administration method for treating various diseases, injection (directly under the skin, muscle, vein or affected area), percutaneous absorption (poultice, application), oral administration and the like have been used. Was. Since these methods can raise the blood concentration of a drug at a stretch, they are effective for improving acute diseases such as infectious diseases and severe symptoms.

For lifestyle-related diseases such as hypertension and diabetes, lifestyle improvement by diet and exercise is effective. However, in a stressful modern society, lifestyle improvement is not easy, and these lifestyle-related diseases are not effective. Prevention, improvement,
There is a need for therapeutics. Chronic diseases such as lifestyle-related diseases require long-term continuous medication, but long-term use of the drug is burdensome for patients, and there are no subjective symptoms or very mild In some cases, there is a problem that it is easy to forget to take the drug, which has been one of the reasons why prevention, improvement, treatment, etc. of these diseases are not sufficiently effective.

[0004] In addition, when the cause of these diseases is hereditary or constitutionally strong, for example, when diabetes is apt to occur in a family, when obesity is strong, or when allergy is predisposed, Since metabolism and enzymatic reactions in the body work in the same direction on a daily basis, it is preferable to keep the effective concentration as constant as possible when using pharmaceuticals, but it is very difficult to achieve this by intermittent drug administration. It is.

On the other hand, as the genetic analysis of humans has progressed recently, genes that cause chronic diseases have been identified, and gene therapy and tailor-made medical treatment based on them have been studied. There are many uncertainties in terms of both efficacy and safety, and it has not yet been completed. Also,
One of the conventional techniques using GMMs is to orally administer GMMs encapsulated in microcapsules to remove unwanted chemicals or amino acids caused by the disease, and use enzymes produced by the microorganisms. Attempts to remove toxins and the like in the body have been reported (see Japanese Patent Application Laid-Open No. 11-502875). However, in this method, the genetically modified microorganism is held in the microcapsule, and unwanted molecules for removal can enter the microcapsule to decompose and metabolize it, but the microorganism itself is not microscopically. Since it is quickly removed from the body while being encapsulated in the capsule, a sustained effect cannot be obtained.

[0006] So far, no drug has been found that can maintain its efficacy for a long period of time in animals, especially humans.

[0007]

Accordingly, there is a need for a drug which is effective for the prevention, amelioration and / or treatment of the above-mentioned chronic diseases, and which can exert its effects continuously for a long period of time. It is an object of the present invention to develop such a drug or a health food which is highly safe and has such an effect.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that a bioactive substance effective for various diseases such as the above-mentioned chronic diseases by using gene recombination technology. A microorganism imparted with a production ability, in particular, a microorganism transformed with an expression vector containing a bioactive substance gene can produce a bioactive substance that effectively acts as its medicinal effect continuously for a long time in the intestine of animals, particularly humans, It has been found that the burden of daily drug administration can be reduced for chronically ill patients, the effect is equal to or better than drug administration, and furthermore, a long-term preventive effect can be obtained for healthy people . Furthermore, it has been found that by using a microorganism that inhabits the human intestinal tract as a microorganism, particularly a useful microorganism such as a lactic acid bacterium, favorable results can be obtained in both effect and safety. The present invention has been completed based on these various findings.

That is, the present invention provides a method for treating a chronic disease characterized by containing a microorganism having a bioactive substance gene recombined therein and enabling the recombinant microorganism to produce the bioactive substance in the intestinal tract of an animal. It is a prophylactic, ameliorating and / or therapeutic agent (hereinafter also referred to as "the pharmaceutical of the present invention").

[0010] In a preferred embodiment of the present invention, the recombinant microorganism is obtained by transforming a microorganism such as Escherichia coli, enterococci, lactic acid bacteria, lactobacilli and bifidobacteria with an expression vector containing a bioactive substance gene. The disease particularly refers to malignant tumor, diabetes, obesity, allergic disease and the like.

[0011] The present invention also provides a method for treating a chronic disease characterized by comprising a microorganism having a recombinant bioactive substance gene, wherein the recombinant microorganism is capable of producing the bioactive substance in the intestinal tract of an animal. It is also present in health foods for prevention or improvement.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The gene for a physiologically active substance used in the present invention includes all kinds of genes capable of producing a substance useful for maintaining and promoting the health of animals, especially humans. For example, useful enzymes include genes such as amylase, protease, peptidase, lipase, various glucosidases and various kinases. Furthermore, genes such as insulin, interferon, growth hormone, and various peptide hormones such as cholecystokinin are included. Also included are genes such as inhibitors of these various enzymes, and genes such as antigenic proteins such as pathogenic bacteria, viruses and parasites.

As the microorganism used in the present invention, Escherichia coli, enterococci and the like can be used, but it is preferable to use useful microorganisms such as lactic acid bacteria, lactobacilli and bifidobacteria. It has been widely known that inhabitation of useful microorganisms such as lactic acid bacteria in the intestinal tract is effective for health promotion and physical condition improvement, and not only lactic acid bacteria preparations, yogurt and lactic acid bacteria drinks but also oligos effective for the growth of these lactic acid bacteria. Foods containing sugar are marketed as foods for specified health use. Therefore, as a host for producing the bioactive substance,
It is considered preferable from the viewpoint of safety.

The expression of a physiologically active substance in a lactic acid bacterium or the like according to the present invention is carried out by introducing a method of introducing an extracellular secretory vector carrying genes of various physiologically active substances into a useful microorganism such as a lactic acid bacterium, a lactobacillus or a bifidobacterium. A method of recombination into the gene of the microorganism itself and inserting genes of various physiologically active substances can be considered. In particular, in lactic acid bacteria, a secretory vector for lactic acid bacteria using a promoter of the extracellular α-amylase gene derived from Streptococcus and a secretory signal (see Japanese Patent Application Laid-Open No. 9-234078) and a method for expressing a fibronectin-binding protein in lactic acid bacteria (particularly, Opening 2000
-236873) has been reported, and it is possible to perform genetic recombination with lactic acid bacteria and the like by using these techniques.

The expression vector containing the recombinant gene can be introduced into a host, such as a lactic acid bacterium, by a conventional method. For example, electroporation (electroporation)
Method (see JP-A-10-80275) or the like can be used.

The recombinant microorganism used in the present invention is:
The ability to produce the desired substance in the intestinal tract of an animal can be easily inferred from the above-described method for producing a recombinant microorganism, and in particular, a drug resistance gene is allowed to coexist in an expression vector containing a recombinant gene, and these drugs are used together with these drugs. By taking the medicine of the present invention, it is possible to prevent the microorganism from dropping out of the intestinal tract.

The method for producing a genetically modified microorganism contained in the medicament of the present invention is specifically described in the following Examples. For example, in Example 1, pancreatic secretory trypsin inhibitor (PSTI) is used as a physiologically active substance. ) Is disclosed. This is a specific inhibitor of trypsin secreted into the pancreatic juice.It is widely related to the defense response of the body besides trypsin inhibition, and it has been reported that blood levels increase when malignant tumors are present. Therefore, an inhibitory effect on malignant tumors is expected. In addition, it has been reported that it promotes insulin secretion through its action of inhibiting trypsin, which is effective in treating diabetes (Am. J. Anatomy, 18
9, 207-212 (1990)).

Therefore, the production of the pancreatic secretory trypsin inhibitor (PSTI) by lactic acid bacteria in the intestinal tract suppresses malignant tumors, in particular, suppression and prevention of cancer in the digestive tract such as pancreatic cancer and colon cancer, and insulin secretion. It is expected to be effective for the treatment of diabetes or the improvement of QOL of patients by promotion.

PSTI is involved in secretion of various digestive enzymes in the digestive tract (Green GM, Lyman RL. Proc. So
c. Exp. Biol. Med. 1972, 140, 6-12 and Ihse I, Lilj
a P, Landquist I. Scand. J. Gastroenterol 1974, 14,
873-80). It is known that there is an oligopeptide (monitor peptide) secreted from the pancreas in the intestinal tract (Fushiki T. and Iwai K. The FASEB Journa
l 1989, 3, 121-126), this peptide is not subject to digestion by the digestive enzymes because the digestive enzymes prioritize the degradation of the protein in the food when the protein is contained in the ingested food. And promotes secretion of proteolytic enzymes such as pepsin, and suppresses secretion of carbohydrate and lipolytic enzymes such as α-amylase and lipase. vice versa,
If the ingested food does not contain protein, it is degraded by proteolytic enzymes and does not bind to receptors in the intestinal tract, thereby promoting the secretion of carbohydrates and lipolytic enzymes and the secretion of proteolytic enzymes. Is suppressed.

Since PSTI inhibits the action of proteolytic enzymes such as trypsin, the degradation of the monitor peptide is inhibited. For this reason, by the above mechanism, in the case of obesity, diabetes, etc., the digestion and absorption of carbohydrates and lipids are suppressed,
The protein is fully digested and absorbed, eliminating the need for excessive dietary restrictions. Therefore, it is effective not only for relieving patients from the stress of dietary restriction, but also for preventing lifestyle-related diseases of young people who frequently eat modern high-fat, high-calorie fast foods and the like.

Furthermore, the intestinal tract is an important site in the body's immune system, and the production of biologically active substances having an immunostimulatory effect, such as roundworm-derived proteins and milk IgG, in the intestinal tract is caused by pollen allergy or atopic allergy. It is effective for prevention, improvement and / or treatment of allergic diseases such as dermatitis.

Allergic diseases such as cedar pollinosis have rapidly increased since the 1980s, but in recent years, the relationship between parasite infection and allergy has been renewed.
The hyper-IgE level seen during parasite infection is due to an increase in non-specific IgE that does not react with other antigens, including parasite antigens.This increase in non-specific IgE is due to specific IgE bound to other antigens. Inhibits binding to mast cells, etc., and suppresses allergic reactions. Research is currently being conducted to apply this effect to the treatment of allergic diseases, but it is considered that it is extremely difficult to treat allergic diseases by actually infecting parasites due to the problem of parasitic infections.

Among the parasites, parasite antigens of a kind that infests the intestinal tract, such as human roundworm and gooseworm, are excretions and secretions of the parasite, and cell-free extracts of the parasite itself. It has been confirmed that a specific fraction has high antigen activity. It is known that such a parasite antigen mainly acts on the living body from the inner surface of the intestine to cause the above-mentioned immune reaction. It has been reported that the characteristic phenomenon at the time of parasite infection is caused by a substance called neutrophil chemotactic factor (NCF) produced by the parasite to escape from the host immune system (J. Parasitol) 1986; 72 (2): 315-2
0, Mol. Immunol 1993; 30 (14): 1315-20).

By using a recombinant technique, a microorganism such as lactic acid bacterium, which is retained in the intestinal tract, has the ability to produce the roundworm-derived antigen and NCF. And produce the same immune response as in roundworm infection. By such an immune response, various allergic diseases can be treated and prevented without being exposed to the risk of parasite infection.

In addition, against bacterial infection of the human body or animals,
In order to produce an antigen capable of inducing an immune response in the body, a method for producing a vaccine comprising a non-pathogenic microorganism expressing a recombinant gene has been reported (Japanese Unexamined Patent Publication (Kokai) No. Heisei 9 (1999)).
6-165688 gazette), but also to improve chronic diseases by supplying a physiologically active substance as described above for a long time,
It does not suggest them. The present invention is not directed to the production of immune antibodies against such foreign antigens, but rather to a specific physiological mechanism that works on the advanced regulatory mechanisms of the immune system of animals, particularly humans, to maintain its homeostasis and prevent the immune system from over-reacting. It is intended to prevent chronic diseases from occurring by supplying the active substance for a long time and stably.

The microorganisms contained in the medicament or health food of the present invention can be cultured by selecting a suitable culture method depending on the species of the host used. For example, in the case of a lactic acid bacterium, static culture is preferably performed at a temperature suitable for the host using a milk or skim milk medium, a commercially available M17 medium, a Lactobacillus MRS medium, or the like, but is not particularly limited. Also,
The microorganisms can be collected from the culture solution by a commonly used method, that is, centrifugation or filtration. Furthermore, when the fermented liquid itself containing the target microorganism such as yogurt can be ingested as food or drink, it is not necessary to collect the microorganism.

The present invention also includes a food or drink (health food) containing the genetically modified microorganism. Even when the food or drink is ingested, the effect of ameliorating a chronic disease can be obtained in the same manner as in the case of the above-mentioned pharmaceuticals, and thus it can be used for preventing lifestyle-related diseases and the like, that is, as a health food.

There are no particular restrictions on the form used as food or drink, and there are, for example, drinks, solids, jelly-like foods and the like, and solids include any of powders, granules, tablets and capsules as preparations. Also processed into such a form is included. In addition, the genetically modified microorganism can be added to noodles such as udon, buckwheat, etc., cookies, biscuits, candies, breads, cakes and other foods, and also to soft drinks, lactic acid drinks and other various drinks.

The medicament or health food of the present invention can be administered orally. The dose varies depending on the recombinant gene expression ability of the microorganism, but is generally 10 ⁶ to 10 ¹⁰ microorganisms / mL.
The concentration is 1 to 100 mL / day. Preferably, the concentration is 10 ⁷ to 10 ⁸ microorganisms / mL and ⁵ to 10 mL / day.

In formulating the pharmaceutical of the present invention, lactic acid bacteria expressing a physiologically active substance are mixed with a pharmaceutically acceptable liquid or solid carrier, and if necessary, a solvent,
A dispersant, emulsifier, buffer, stabilizer, excipient, binder, disintegrant, and the like can be added to the composition and used in the form of tablets, granules, powders, powders, capsules, and the like. A particularly preferred oral formulation is an enteric coating. This formulation dissolves in the intestine of the ingested animal, but is resistant to acids and enzymes in the stomach. In addition, the retention days and activity of the microorganism in the intestinal tract,
Since the influence of the diet and the intestinal flora is great, it is possible to select the optimal administration interval and administration method according to each individual.

[0031]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

(Example 1) Expression of human PSTI in the intestinal tract cDNA of secretory trypsin inhibitor derived from human pancreas
Has already been cloned (Yamamoto T, Nakamura
Y, Nishide J, Emi M, Ogawa M, Mori T, Matsubara K.
Biochem Biophys Res Commun 1985 132: 605-12), SEQ ID NO: 1 shows its nucleotide sequence, and SEQ ID NO: 2 shows its amino acid sequence. Using this as a clue, oligonucleotide primers having the nucleotide sequences shown in SEQ ID NOs: 3 and 4 were prepared, PCR was performed using the human PSTI cDNA as a template, and the amplified DNA fragment was used as an E. coli expression vector pGEX-3X.
(Amersham Pharmacia Biotech) at the SmaI cleavage site. A recombinant plasmid pGPSTI in which a cDNA fragment of PSTI is inserted in the forward direction with respect to the tac promoter of Escherichia coli is selected, and Escherichia coli JM109 is transformed to obtain recombinant Escherichia coli JM109 (pGPSTI) having PSTI-producing ability.

This recombinant Escherichia coli JM109 (pGPSTI) was cultured in an LB medium, the culture was centrifuged, the cells were collected, and then resuspended in a phosphate buffered saline (10%). ⁸ / mL) together with the marker antibiotic ampicillin in mice (ddy
Strain, male, 6 weeks old) and reared for 14 days. The mouse was a normal diet having the composition shown in Table 1, a normal diet + bacterial suspension,
High fat diet, high fat diet + bacterial suspension were divided into 10 groups, and rearing conditions were set in individual plastic gauges, 23 ± 2
° C, 12 hours sunshine, feed and water (tap water) are available ad libitum.

[Table 1] Composition of feed

^* : In the group to which the bacterial suspension was added, the water was reduced by 10 mL, and the suspension was added by 10 mL. During the breeding period, the mice were weighed. After breeding, blood was collected by cardiac puncture, and total cholesterol (cholesterol E test Wako (Wako Pure Chemical Industries)) and neutral fat (Nescort + G kit (Nihon Shoji)) were measured. The results are shown in Tables 2 and 3. As a result, there is no difference between the normal diet and the normal diet + germ suspension, but there is a difference between the high-fat diet and the high-fat diet + germ suspension. From these results, it is understood that administration of a recombinant microorganism having a PSTI-producing ability is effective for preventing obesity and preventing lifestyle-related diseases.

[Table 2] Effect of bacterial suspension on weight change of mice

[Table 3] Effect of bacterial suspension on total serum cholesterol in mice

Example 2 Intestinal Expression of Human Roundworm-Induced Neutrophil-Inducing Factor Neutrophil-Inducing Factor (NCF) Gene from Human Roundworm , And measure the IgE concentration in the blood to confirm that hyper-IgE is present.

The NCF derived from human roundworm is the DiNCF gene sequence (Owhash) derived from Dirofilaria immitis shown in SEQ ID NO: 5.
i M., Futaki S., Kitagawa K., Horii Y., Maruyama
H., Hayashi H., Nawa Y., Mol. Immunol. 1993, 30, 1
315-20), using primers consisting of the nucleotide sequences shown in SEQ ID NOs: 7 and 8 to form Ascaris iumbricoides
Approximately 1.1 kb from the cell-free extract of
Obtain a PCR product. This PCR product was cloned into pBR322, and further, a vector pGEX-3X for protein expression in E. coli.
To create pGNCF. Escherichia coli JM109 was transformed using this expression type plasmid according to a conventional method, and the resulting transformant Escherichia coli JM109 (pGNCF) was cultured in an LB medium, proteins were extracted from the cells, and SDS-PAGE was performed.
As a result, a clone forming a 14-kilodalton band of the same size as NCF is obtained. Extract pGNCF from this clone, cut out the SmaI-BamHI fragment containing the NCF gene,
After blunting using BluntingKit (manufactured by Takara Shuzo Co., Ltd.), pSECE1, which is a broad host vector for lactic acid bacteria (JP-A-9-234078)
Publication No.) Insertion into EcoRV site. This insert is amy
Lactobacillus casei was transformed by electroporation (see Japanese Patent Application Laid-Open No. 10-80275) with the recombinant plasmid pSENCF inserted in the forward direction relative to the A promoter, and M17 plates containing 25 μg / mL of erythromycin were used. The cells were cultured in a medium, and the grown colonies were further cultured in a test tube in an M17 liquid medium containing 1% lactose.
Remove the cells from each cultured test tube by centrifugation,
A clone showing a protein band of the same size as NCF in the culture solution is obtained. The obtained NCF-producing clone is further cultured to obtain a culture solution and a cell suspension.

The culture was centrifuged to collect the cells, and the cell suspension (10 ⁸ / mL) resuspended in phosphate buffered saline was added to 10, 20, 30 mL of the feed weight per 100 g. And mouse with BALB / c strain, male, 5 weeks old, 23 ±
2 g) of bait (MF, manufactured by Oriental Yeast Co., Ltd.)
Raise for 10 days. The breeding condition is a group of 10 animals, put in individual plastic gauges, 23 ± 2 ° C, 12 hours of sunshine, and free access to feed and water (tap water).

Under the same conditions, 5 μg of dinitrophenylated roundworm extract (Cosmo Bio) and 2 mg of aluminum hydroxide gel (Cosmo Bio) were added at the start of breeding.
A solution dissolved in 2 mL of phosphate buffered saline is intraperitoneally administered to a mouse and immunized to prepare a high-concentration IgE group.

On the 10th day, blood was collected by cardiac puncture,
Also, the intestinal tract is collected by dissection and homogenized in a buffer to obtain an intestinal contents sample. For the blood, after separating serum, the results of measuring the IgE concentration in the blood using an ELISA Mouse IgE Kit (Morinaga Mouse IgE Measurement Kit, manufactured by Seikagaku Corporation) are shown in Table 4. As a result, IgE in blood shows a significantly higher value in the group to which the bacterial cell suspension was given than in the group to which no bacterial suspension was given.

[Table 4] Total IgE concentration in blood of mice (ng / mL)

The intestinal contents sample was subjected to DN extraction by hot water extraction.
After extracting A, as a result of performing PCR using the primers described above,
Approximately 2 samples of the intestinal contents of the group given the cell suspension
Obtain a 1.1 kb PCR product. This is considered to be derived from the NCF-producing clone. These results suggest that NCF-producing cloned lactic acid bacteria can produce NCF in the intestinal tract and thereby maintain a high IgE state.

Further, in order to confirm the effect on allergy using NCF-producing clones, 30 mL of the cell suspension (10 ⁸ / mL) was mixed with 100 g of the feed weight, and mice (ddy, male, 8 Weekly, 33 ± 2 g) and fed (MF, manufactured by Oriental Yeast Co., Ltd.) and bred for 10 days. Rearing conditions are the same as those described above.

After breeding, 10 μL of mouse monoclonal anti-dinitrophenyl group IgE (manufactured by Seikagaku Corporation) diluted 5000 times with phosphate buffered saline is intradermally administered to both auricles by intradermal injection. Thereafter, the animals are reared for another 2 days, and 0.25 mL of a solution of 0.25 mg of dinitrophenylated bovine serum albumin dissolved in 0.5% Evans blue is administered through the tail vein to cause a passive cutaneous anaphylactic reaction (PCA). A group to which ketofinal (manufactured by Sando Pharmaceutical) was orally administered (1 mL / kg) is used as a comparison. Thereafter, the amount of pigment in both ears was extracted and quantified, and a comparison value was determined with the control group as 100. The results are shown in Table 5. From these results, an effect of suppressing the passive cutaneous anaphylactic reaction (PCA) is seen.

[Table 5] Inhibitory effect on mouse PCA reaction

Further, in order to confirm the effect on contact dermatitis, the abdomen was shaved, and a 2.5% dinitrofluorobenzeneethanol solution (DNFB) was applied to immunize. The animals were reared under the same conditions as described above, the back hair was cut after 5 days and 10 days, 20% of 1% DNFB was applied, and the magnitude of inflammation was measured with a gauge. Table 6 shows the results. From these results, in the group to which the cell suspension was administered, inflammation was suppressed after 5 days as compared to the control group, but no difference was observed in the magnitude of inflammation after 10 days. This suggests that the NCF-producing clone has no effect on the secondary immune response in the immune response.

[Table 6] Average diameter of skin inflammation in mice (mm)

From the above results, it can be seen that the mice to which the lactic acid bacterium suspension producing NCF was orally administered had an increased amount of non-specific IgE and a suppressed specific immune reaction.

[0051]

By taking the pharmaceutical or health food of the present invention, a continuous and high effect of a physiologically active substance absorbed from the intestinal tract or acting in the intestinal tract can be obtained. By the effect of such a physiologically active substance, prevention, improvement and / or treatment of chronic diseases such as lifestyle-related diseases and allergies can be extremely effectively achieved, and the burden of patients on continuous medication can be reduced. In addition, it is possible for general healthy persons and persons with such signs to easily eat and drink in the form of health food.

[0052]

[Sequence List] SEQUENCE LISTING <110> SANPO KK <120> A preventive and / or a therapeutic agent for chronic desease <130> P6847GN <140> <141> <160> 8 <170> PatentIn Ver. 2.1 <210> 1 <211> 368 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (61) .. (297) <400> 1 gaagagacgt ggtaagtgcg gtgcagtttt caactgacct ctggacgcag aacttcagcc 60 atg aag gta aca ggc atc ttt ctc agt gcc ttg gcc ctg ttg agt 108 Met Lys Val Thr Gly Ile Phe Leu Leu Ser Ala Leu Ala Leu Leu Ser 1 5 10 15 cta tct ggt aac act gga gct gac tcc ctg gga aga gag gcc aaa tgt 156 Leu Ser Gly Asn Thr Gly Ala Asp Ser Leu Gly Arg Glu Ala Lys Cys 20 25 30 tac aat gaa ctt aat gga tgc acc aag ata tat gac cct gtc tgt ggg 204 Tyr Asn Glu Leu Asn Gly Cys Thr Lys Ile Tyr Asp Pro Val Cys Gly 35 40 45 act gat gga aat act tat ccc aat gaa tgc gtg tta tgt ttt gaa ggt 252 Thr Asp Gly Asn Thr Tyr Pro Asn Glu Cys Val Leu Cys Phe Glu Gly 50 55 60 cgg aaa cgc cag act tct atc ctc att caa aaa tct ggg cct tgc 297 Arg Lys Arg Gln Thr Ser Ile Leu Ile Gln L ys Ser Gly Pro Cys 65 70 75 tgagaaccaa ggttttgaaa tcccatcagg tcaccgcgag gcctattgtt gaataaatgt 357 atctgaatat c 368 <210> 2 <211> 79 <212> PRT <213> Homo sapiens <400> 2 Met Lys Val Thr Gly Ile Phe Leu Leu Ala Leu Leu Ser 1 5 10 15 Leu Ser Gly Asn Thr Gly Ala Asp Ser Leu Gly Arg Glu Ala Lys Cys 20 25 30 Tyr Asn Glu Leu Asn Gly Cys Thr Lys Ile Tyr Asp Pro Val Cys Gly 35 40 45 Thr Asp Gly Asn Thr Tyr Pro Asn Glu Cys Val Leu Cys Phe Glu Gly 50 55 60 Arg Lys Arg Gln Thr Ser Ile Leu Ile Gln Lys Ser Gly Pro Cys 65 70 75 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 3 atgaaggtaa caggcatctt 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 4 tcagcaaggc ccagattttt g 21 <210> 5 <211> 993 <212> DNA <213> Dirofilaria immitis <220> <221> CDS <222> (1) .. (993) <400> 5 att ttt gat tat ttc gaa agt ttg aca ggt gat aaa aag aaa aag gct 48 Ile Phe Asp Tyr P he Glu Ser Leu Thr Gly Asp Lys Lys Lys Lys Ala 1 5 10 15 gca gaa gaa ctt caa caa ggt tgc tta atg gct ctc agt gaa atc att 96 Ala Glu Glu Glu Leu Gln Gln Gly Cys Leu Met Ala Leu Ser Glu Ile Ile 20 25 30 ggt aat gaa aag atg ctt atg ttg aaa gag att aaa gat tca ggc gct 144 Gly Asn Glu Lys Met Leu Met Leu Lys Glu Ile Lys Asp Ser Gly Ala 35 40 45 gat cca gaa caa atc gaa gat atg ttg aaa ctt gtt gac aaa gaa 192 Asp Pro Glu Gln Ile Glu Asp Met Leu Lys Leu Val Val Asp Lys Glu 50 55 60 aag aag aaa aga att gat gaa tat cct cct gta tgc cgt aaa att tat 240 Lys Lys Lys Arg Ile Asp Glu Tyr Pro Pro Val Cys Arg Lys Ile Tyr 65 70 75 80 gcg gca atg aat gaa cgg cgt aag cgg aat gat cat aat tta gaa agc 288 Ala Ala Met Asn Glu Arg Arg Lys Arg Asn Asp His Asn Leu Glu Ser 85 90 95 tat ttt cag acg tat ctg agc tgg ctc aca gat gct caa aaa gat gaa 336 Tyr Phe Gln Thr Tyr Leu Ser Trp Leu Thr Asp Ala Gln Lys Asp Glu 100 105 110 att aaa aaa atg aaa gaa gaa gga aaa tcg aaa atg gat atat caa Ile Lys Lys Met Lys Glu Glu Gly Lys Ser Lys Met Asp Ile Gln Lys 115 120 125 aaa att ttt gat tat ttc gaa agt ttg aca ggt gat aaa aag aaa aag 432 Lys Ile Phe Asp Tyr Phe Glu Ser Leu Thrhr Gly Asp Lys Lys Lys Lys 130 135 140 gct gca gaa gaa ctt caa gaa ggc tgc aga atg gct ctg aga gaa att 480 Ala Ala Glu Glu Leu Gln Glu Gly Cys Arg Met Ala Leu Arg Glu Ile 145 150 155 160 gtt ggt gaa gag aag tgg act gta atg agg cag tca gca 528 Val Gly Glu Glu Lys Trp Thr Val Met Arg Gln Met Lys Asp Ser Ala 165 170 175 act cca aag gaa cta agc atg aaa gtt gaa gag atg ttc aaa gat gtc 576 Thr Pro Lys Glu Leu Ser Met Lys Val Glu Glu Met Phe Lys Asp Val 180 185 190 gtt gac aaa gat aaa aag gaa aaa att gat gaa tat gct cct gta tgc 624 Val Asp Lys Asp Lys Lys Glu Lys Ile Asp Glu Tyr Ala Pro Val Cys 195 200 205 cgt aaa atc ttt gcg gtg ata cat gaa agg cgt aag agg aat gat cat 672 Arg Lys Ile Phe Ala Val Ile His Glu Arg Arg Lys Arg Asn Asp His 210 215 220 aat tta gaa agc tat ttt caa acg tat ctg agc tgg ctc acg gat gct 720 Asn Leu GluSer Tyr Phe Gln Thr Tyr Leu Ser Trp Leu Thr Asp Ala 225 230 235 240 caa aaa gat gaa att aaa aaa atg aaa gaa gaa gga aaa tcg aaa atg 768 Gln Lys Asp Glu Ile Lys Lys Met Lys Glu Glu Gly Lys Ser Lys Met 245 250 255 gat att caa aaa aaa att ttt gat tat ttc gaa agt ttg aca ggt gat 816 Asp Ile Gln Lys Lys Ile Phe Asp Tyr Phe Glu Ser Leu Thr Gly Asp 260 265 270 270 aaa aag aaa aag gct gca gaa caa cat caa ggt tgc tta atg gct 864 Lys Lys Lys Lys Ala Ala Glu Glu Leu Gln Gln Gly Cys Leu Met Ala 275 280 285 ctc agt gaa atc att ggt aat gaa aag atg ctt atg ttg aaa gag att 912 Leu Ser Glu Ile Lys Met Leu Met Leu Lys Glu Ile 290 295 300 aaa gat tca ggc gct gat cca gaa caa atc aga atg aaa gtc gaa gat 960 Lys Asp Ser Gly Ala Asp Pro Glu Gln Ile Arg Met Lys Val Glu Asp 305 310 315 320 atg aaa ctt gtc gtt aac aaa gaa aaa agc 993 Met Leu Lys Leu Val Val Asn Lys Glu Lys Ser 325 330 <210> 6 <211> 331 <212> PRT <213> Dirofilaria immitis <400> 6 Ile Phe Asp Tyr Phe Glu Ser Leu Thr Gly Asp L ys Lys Lys Lys Ala 1 5 10 15 Ala Glu Glu Leu Gln Gln Gly Cys Leu Met Ala Leu Ser Glu Ile Ile 20 25 30 Gly Asn Glu Lys Met Leu Met Leu Lys Glu Ile Lys Asp Ser Gly Ala 35 40 45 Asp Pro Glu Gln Ile Glu Asp Met Leu Lys Leu Val Val Asp Lys Glu 50 55 60 Lys Lys Lys Arg Ile Asp Glu Tyr Pro Pro Val Cys Arg Lys Ile Tyr 65 70 75 80 Ala Ala Met Asn Glu Arg Arg Lys Arg Asn Asp His Asn Leu Glu Ser 85 90 95 Tyr Phe Gln Thr Tyr Leu Ser Trp Leu Thr Asp Ala Gln Lys Asp Glu 100 105 110 Ile Lys Lys Met Lys Glu Glu Gly Lys Ser Lys Met Asp Ile Gln Lys 115 120 125 Lys Ile Phe Asp Tyr Phe Glu Ser Leu Thr Gly Asp Lys Lys Lys Lys 130 135 140 Ala Ala Glu Glu Leu Gln Glu Gly Cys Arg Met Ala Leu Arg Glu Ile 145 150 155 160 Val Gly Glu Glu Glu Lys Trp Thr Val Met Arg Gln Met Lys Asp Ser Ala 165 170 175 Thr Pro Lys Glu Leu Ser Met Lys Val Glu Glu Met Phe Lys Asp Val 180 185 190 Val Asp Lys Asp Lys Lys Glu Lys Ile Asp Glu Tyr Ala Pro Val Cys 195 200 205 Arg Lys Ile Phe Ala Val Ile His Glu Arg Arg Lys Arg Asn Asp His 210 215 220 Asn Leu Glu Ser Tyr Phe Gln Thr Tyr Leu Ser Trp Leu Thr Asp Ala 225 230 235 240 Gln Lys Asp Glu Ile Lys Lys Met Lys Glu Glu Gly Lys Ser Lys Met 245 250 255 Asp Ile Gln Lys Lys Ile Phe Asp Tyr Phe Glu Ser Leu Thr Gly Asp 260 265 270 Lys Lys Lys Lys Ala Ala Glu Glu Leu Gln Gln Gly Cys Leu Met Ala 275 280 285 285 Leu Ser Glu Ile Ile Gly Asn Glu Lys Met Leu Met Leu Lys Glu Ile 290 295 300 Lys Asp Ser Gly Ala Asp Pro Glu Gln Ile Arg Met Lys Val Glu Asp 305 310 315 320 Met Leu Lys Leu Val Val Asn Lys Glu Lys Ser 325 330 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: PCR primer <400> 7 atttttgatt atttcgaaag tttg 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer < 400> 8 tctgatttgt tctggatcag cgcc 24

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 37/08 A61P 37/08 C12N 1/20 C12N 1/20 E 1/21 1/21 15/09 ZNA (C12N 1/20 E // (C12N 1/20 C12R 1:19) C12R 1:19) (C12N 1/20 E (C12N 1/20 C12R 1:01) C12R 1:01) (C12N 1/21 ( C12N 1/21 C12R 1:19) C12R 1:19) (C12N 1/21 (C12N 1/21 C12R 1:01) C12R 1:01) C12N 15/00 ZNAA F term (reference) 4B018 LB01 LB02 LB08 MD20 MD85 MD86 ME01 ME03 ME04 ME07 ME08 4B024 AA01 AA05 BA21 BA80 CA04 DA05 DA06 EA04 GA11 GA19 HA01 HA12 4B065 AA01X AA26X AA90Y AB01 AC14 BA03 BA25 CA24 CA41 CA44 4C087 AA01 AA02 BC34 BC56 BC60 BC61 CA12 NA13

Claims (10)

[Claims] 1. A method for preventing and improving chronic diseases, comprising a microorganism having a recombinant bioactive substance gene, wherein said recombinant microorganism is capable of producing said bioactive substance in the intestinal tract of an animal. And / or therapeutic agents. 2. The preventive, ameliorating and / or therapeutic agent according to claim 1, wherein the physiologically active substance is a pancreatic secretory trypsin inhibitor or a neutrophil chemotactic factor. 3. The preventive, ameliorating and / or therapeutic agent according to claim 1, wherein the recombinant microorganism has been transformed with an expression vector containing the bioactive substance gene. 4. The preventive, ameliorating and / or therapeutic agent according to claim 1, wherein the recombinant microorganism is at least one of Escherichia coli, enterococci, lactic acid bacteria, lactobacilli and bifidobacteria. 5. The method according to claim 1, wherein the chronic disease is at least one of malignant tumor, diabetes, obesity and allergic disease.
The preventive, ameliorating and / or therapeutic agent according to any one of claims 4 to 4. 6. A method for preventing or ameliorating a chronic disease, comprising a microorganism having a bioactive substance gene recombined therein, wherein said recombinant microorganism is capable of producing said bioactive substance in the intestinal tract of an animal. healthy food. 7. The health food according to claim 6, wherein the physiologically active substance is a pancreatic secretory trypsin inhibitor or a neutrophil chemotactic factor. 8. The health food according to claim 6, wherein the recombinant microorganism has been transformed with an expression vector containing the bioactive substance gene. 9. The health food according to claim 6, wherein the recombinant microorganism is at least one of Escherichia coli, enterococci, lactic acid bacteria, lactobacilli and bifidobacteria. 10. The chronic disease is at least one of malignant tumor, diabetes, obesity and allergic disease.
9. The health food according to any one of items 9.